The present invention relates to a highly sensitive method for electrochemically detecting a DNA using a unique combination of compounds as a mixed intercalator and a detection kit using same.
DNA chips have been widely used in gene and molecular biology researches such as the measurement of RNA expression in a large scale, detection of mutant genome DNAs, gene diagnosis, pharmacogenomics and medicine, as they can detect RNAs or DNAs contained in a sample much more efficiently than the conventional Southern blot or Northern blot method.
Generally, DNA chips detect a target DNA, for example, by way of accumulating hundreds of thousands of probe DNA fragments, each having a specified base sequence, on a very small chip surface, contacting the probe DNA fragments with a single strand of the target DNA labeled with a fluorescent material to induce hybridization, and identifying the hybridized DNA by laser irradiation.
However, the above method has the disadvantages that it requires the use of an expensive optical apparatus including a laser scanner and the cost of fluorescent labeling is high. Further, it is difficult to quantitatively determine the amount of the target DNA in a sample from the luminescent intensity.
Accordingly, there have been numerous efforts to solve the above-mentioned problems. For instance, Clinical Microsensors Inc. suggests a method of detecting a DNA by binding a redox-active material, e.g., a transition metal complex, on a selected site of a single-stranded probe DNA, bringing a single-stranded target DNA into contact with the resulting probe DNA to induce hybridization, and measuring the change in the electron transporting rate attributable to the hybridization. In addition, Japanese Patent Publication No. 2000-125865 provides a method of detecting a gene of a specimen DNA by allowing a single-stranded sample DNA to interact with a single strand probe DNA immobilized on an electrode surface in the presence of an electrochemically active intercalator to form a hybridized DNA carrying the intercalator, followed by determining the current which flows through the intercalator.
These methods, however, still exhibit a limited sensitivity for quantitative DNA detection, and thus, there has existed a need to develop a DNA detection method having a higher sensitivity.
Accordingly, it is an object of the present invention to provide an improved method for detecting a DNA with a high sensitivity.
It is another object of the present invention to provide a novel intercalator and a detection kit which are suitable for practicing the method.
In accordance with one aspect of the present invention, there is provided a method for detecting a DNA having a specified base sequence (target DNA), which comprises bringing a single strand of the target DNA (target ssDNA) into contact with a single strand probe DNA (probe ssDNA) bonded on the surface of an electrode equipped with an output terminal to form a double strand DNA (dsDNA), adding a mixed intercalator consisting of compounds of formulae (I) and (II) to intercalate the dsDNA therewith, and determining the current generated when a voltage is applied to the electrode.
In accordance with another aspect of the present invention, there is provided an alternative method for detecting a DNA having a specified base sequence (target DNA), which comprises bringing a single strand of the target DNA (target ssDNA) into contact with a single strand probe DNA (probe ssDNA) bonded on the surface of an electrode equipped with an output terminal in the presence of a mixed intercalator consisting of compounds of formulae (I) and (II) to obtain a double strand DNA intercalated by said compounds, and determining the current generated when a voltage is applied to the electrode: 
In accordance with still another aspect of the present invention, there is provided a DNA detection kit used in practicing the method, comprising a DNA sensor having a layer of single strand probe DNAs bonded on the surface of an electrode equipped with an output terminal and a mixed intercalator consisting of compounds of formulae (I) and (II); and the mixed intercalator.